The CDK1 protein kinase is the universal trigger of mitosis. In turn its activity is regulated by protein kinases of the Wee1 family and phosphoprotein phosphatases of the Cdc25 family. Here we propose three specific aims on the cooperative regulation of Wee1A by CDK1; the mechanisms of ultrasensitivity in the regulation of Cdc25C by CDK1; and the regulation of the phosphatases that oppose the phosphorylation of Wee1A and Cdc25C during the transition from interphase into M-phase. 1. For Wee1A, phosphorylation promotes CDK1 binding, which promotes more Wee1A phosphorylation. What is the mechanism for this? Our working hypothesis is that the p9 Cks2 protein acts as a phosphoepitope-binding subunit of the CDK1 complex, and is responsible for the increased affinity of CDK1-cyclin B1 for phospho-Wee1A compared to dephospho-Wee1. We plan to test this hypothesis through reconstitution, immunodepletion, and mutagenesis approaches. 2. Does Cdc25C phosphorylation promote CDK1-cyclin B1 binding, and if so does that explain Cdc25C's high intrinsic ultrasensitivity? The hyperphosphorylation of Cdc25C is a highly ultrasensitive function of the level of CDK1-cyclin B1 activity, well-approximated by a Hill function with a Hill coefficient of 11. Much of this ultrasensitivity can be seen in reconstituted systems composed of purified CDK1-cyclin B1 and Cdc25C. We plan to test the hypothesis that phosphorylation-induced CDK1-cyclin B1 binding is responsible for this intrinsic ultrasensitivity. 3. What phosphatases dephosphorylate Wee1A and Cdc25C during the transition into mitosis? Are they regulated? Does their regulation account for (or contribute to) the extrinsic ultrasensitivity of the Wee1A and Cdc25C responses? Recent work using model CDK1 substrates has implicated PP1 and/or PP2A-B554 as regulators of protein dephosphorylation during mitotic exit. Here we plan to use a panel of Wee1A and Cdc25C mutants to ask how the rates of dephosphorylation of various sites in these two proteins vary with the activity of CDK1 in Xenopus egg extracts; whether the sites behave differently or similarly to each other; what phosphatases are responsible for their dephosphorylation and whether these phosphatases are regulated in a switch-like, ultrasensitive fashion. PUBLIC HEALTH RELEVANCE: Cdc25 and Wee1 regulate mitosis in (as far as we know) all eukaryotic cells. From a fundamental perspective their own regulation is of interest because (1) mitotic entry is such a dramatic, important cell biological process; (2) the regulation of the Cdc25 and Wee1 has already yielded important general insight into the principles of protein regulation, and promises to continue to do so; (3) pharmacological Wee1 inhibitors hold promise as cancer chemotherapeutic agents.